Leiomyomas are benign uterine tumors which develop from transformation of myometrial cells without a known etiology. Through genomic and proteomic analysis we have identified a number of differentially expressed and regulated genes in leiomyomas as compared to myometrium, with diverse biological functions, including cellular transformation, proliferation, and pro-inflammatory/pro-fibrotic activities such as IL-13 and TGF-2. ZEB1, a transcription factor and major repressor of E-cadherin, which is lost during cellular transition into mesenchymal phenotype, is overexpressed in leiomyomas as compared to myometrium. Based on differential expression, hormonal regulation and regulation by TGF-2 and IL-13, we propose that ZEB1 and ZEB2 also serve as key regulators of myometrial phenotypic transformation into leiomyomas. We further propose that expression and stability of ZEBs are subject to epigenetic and microRNA regulation, respectively. To test the hypothesis that the expression, regulation and function of ZEBs result in development and growth of leiomyoma, we propose the following specific aims. Aim#1 will test the hypothesis that ZEBs and E-cadherin are differentially expressed and regulated in leiomyomas as compared to myometrium, and their expression directly/inversely correlates with the expression of TGF-2 and IL-13 and altered in patients who receive hormonal therapies to suppress leiomyoma growth. Furthermore, the expression profiles of HDAC's 2, 4, and EZH2 in leiomyoma and myometrium from the above cohorts correlate with their role as epigenetic regulators of the expression of ZEBs and miRNAs that target their expression, and are differentially expressed in tissues from African Americans as compared to Caucasians. Aim#2 will test the hypothesis that pro-inflammatory/pro-fibrotic molecules, such as IL-13 and TGF-2 directly regulate the expression of ZEBs in myometrial smooth muscle cells (MSMC) resulting in phenotypic transformation as a consequence of loss of E-cadherin, and their actions are prevented following treatments with GnRHa, RU-486, Depo-Provera and Tranilast, an inhibitor of pro-fibrotic/inflammatory mediators. Further functional activity of ZEBs is altered through treatments with siRNAs and miR-200b/c in MSMC and LSMC thus their downstream target genes, including E-cadherin. Aim#3 will test the hypothesis that ZEBs expression is regulated via epigenetic modifications and by the action of miRNAs in myometrial cells, and alteration in this mechanism results in transformation into leiomyoma cells. This will be tested by assessing the patterns of expression of HDACs and EZH2, as well as miRNAs that target their expression in myometrial and leiomyoma cells;following treatments with IL-13 and TGF-2, alone and in combination, and following treatment with Tranilast, miRNAs gain-of functions and inhibitors of HDACs and CpG methylation, respectively. To achieve these Aims, we will use leiomyoma and matched myometrium and utilize a combination of biochemical, molecular and cell biological approaches. We anticipate that this proposed research will lead to further identification of a direct role of pro-fibrotic/inflammatory derived mediators in myometrial cellular transformation into leiomyoma cells and help to work toward the overall development of a novel therapeutic approach to prevent the growth and specifically treat leiomyomas. PUBLIC HEALTH RELEVANCE: Fibroids are benign uterine tumors estimated to develop in 70% of women, more specifically among African Americans during their reproductive years. Symptomatic fibroids cause chronic pelvic pain and abnormal uterine bleeding which are the main indications for women undergoing hysterectomy. This proposal will investigate how fibroids are developed and grow, and ways to prevent them, including their symptoms.